Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 8 de 8
Filtrar
1.
Circulation ; 148(1): 47-67, 2023 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-37199168

RESUMO

BACKGROUND: Activation of vascular smooth muscle cell (VSMC) inflammation is vital to initiate vascular disease. The role of human-specific long noncoding RNAs in VSMC inflammation is poorly understood. METHODS: Bulk RNA sequencing in differentiated human VSMCs revealed a novel human-specific long noncoding RNA called inflammatory MKL1 (megakaryoblastic leukemia 1) interacting long noncoding RNA (INKILN). INKILN expression was assessed in multiple in vitro and ex vivo models of VSMC phenotypic modulation as well as human atherosclerosis and abdominal aortic aneurysm. The transcriptional regulation of INKILN was verified through luciferase reporter and chromatin immunoprecipitation assays. Loss-of-function and gain-of-function studies and multiple RNA-protein and protein-protein interaction assays were used to uncover a mechanistic role of INKILN in the VSMC proinflammatory gene program. Bacterial artificial chromosome transgenic mice were used to study INKILN expression and function in ligation injury-induced neointimal formation. RESULTS: INKILN expression is downregulated in contractile VSMCs and induced in human atherosclerosis and abdominal aortic aneurysm. INKILN is transcriptionally activated by the p65 pathway, partially through a predicted NF-κB (nuclear factor kappa B) site within its proximal promoter. INKILN activates proinflammatory gene expression in cultured human VSMCs and ex vivo cultured vessels. INKILN physically interacts with and stabilizes MKL1, a key activator of VSMC inflammation through the p65/NF-κB pathway. INKILN depletion blocks interleukin-1ß-induced nuclear localization of both p65 and MKL1. Knockdown of INKILN abolishes the physical interaction between p65 and MKL1 and the luciferase activity of an NF-κB reporter. Furthermore, INKILN knockdown enhances MKL1 ubiquitination through reduced physical interaction with the deubiquitinating enzyme USP10 (ubiquitin-specific peptidase 10). INKILN is induced in injured carotid arteries and exacerbates ligation injury-induced neointimal formation in bacterial artificial chromosome transgenic mice. CONCLUSIONS: These findings elucidate an important pathway of VSMC inflammation involving an INKILN/MKL1/USP10 regulatory axis. Human bacterial artificial chromosome transgenic mice offer a novel and physiologically relevant approach for investigating human-specific long noncoding RNAs under vascular disease conditions.


Assuntos
Aneurisma da Aorta Abdominal , RNA Longo não Codificante , Animais , Humanos , Camundongos , Aneurisma da Aorta Abdominal/metabolismo , Proliferação de Células , Células Cultivadas , Inflamação/genética , Inflamação/metabolismo , Luciferases/metabolismo , Camundongos Transgênicos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , NF-kappa B/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Ubiquitina Tiolesterase/metabolismo
2.
Circ Res ; 125(5): 535-551, 2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31339449

RESUMO

RATIONALE: In response to blood vessel wall injury, aberrant proliferation of vascular smooth muscle cells (SMCs) causes pathological remodeling. However, the controlling mechanisms are not completely understood. OBJECTIVE: We recently showed that the human long noncoding RNA, SMILR, promotes vascular SMCs proliferation by a hitherto unknown mechanism. Here, we assess the therapeutic potential of SMILR inhibition and detail the molecular mechanism of action. METHODS AND RESULTS: We used deep RNA-sequencing of human saphenous vein SMCs stimulated with IL (interleukin)-1α and PDGF (platelet-derived growth factor)-BB with SMILR knockdown (siRNA) or overexpression (lentivirus), to identify SMILR-regulated genes. This revealed a SMILR-dependent network essential for cell cycle progression. In particular, we found using the fluorescent ubiquitination-based cell cycle indicator viral system that SMILR regulates the late mitotic phase of the cell cycle and cytokinesis with SMILR knockdown resulting in ≈10% increase in binucleated cells. SMILR pulldowns further revealed its potential molecular mechanism, which involves an interaction with the mRNA of the late mitotic protein CENPF (centromere protein F) and the regulatory Staufen1 RNA-binding protein. SMILR and this downstream axis were also found to be activated in the human ex vivo vein graft pathological model and in primary human coronary artery SMCs and atherosclerotic plaques obtained at carotid endarterectomy. Finally, to assess the therapeutic potential of SMILR, we used a novel siRNA approach in the ex vivo vein graft model (within the 30 minutes clinical time frame that would occur between harvest and implant) to assess the reduction of proliferation by EdU incorporation. SMILR knockdown led to a marked decrease in proliferation from ≈29% in controls to ≈5% with SMILR depletion. CONCLUSIONS: Collectively, we demonstrate that SMILR is a critical mediator of vascular SMC proliferation via direct regulation of mitotic progression. Our data further reveal a potential SMILR-targeting intervention to limit atherogenesis and adverse vascular remodeling.


Assuntos
Proliferação de Células/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Proteínas dos Microfilamentos/metabolismo , Mitose/fisiologia , Músculo Liso Vascular/metabolismo , RNA Longo não Codificante/biossíntese , Remodelação Vascular/fisiologia , Ciclo Celular/fisiologia , Células Cultivadas , Proteínas Cromossômicas não Histona/genética , Humanos , Proteínas dos Microfilamentos/genética , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Técnicas de Cultura de Órgãos , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Veia Safena/citologia , Veia Safena/metabolismo
3.
Arterioscler Thromb Vasc Biol ; 40(3): 697-713, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31826651

RESUMO

OBJECTIVE: Long noncoding RNAs (lncRNAs) are an emergent class of molecules with diverse functional roles, widely expressed in human physiology and disease. Although some lncRNAs have been identified in cardiovascular disease, their potential as novel targets in the prevention of atherosclerosis is unknown. We set out to discover important lncRNAs in unstable plaque and gain insight into their functional relevance. Approach and Results: Analysis of RNA sequencing previously performed on stable and unstable atherosclerotic plaque identified a panel of 47 differentially regulated lncRNAs. We focused on LINC01272, a lncRNA upregulated in unstable plaque previously detected in inflammatory bowel disease, which we termed PELATON (plaque enriched lncRNA in atherosclerotic and inflammatory bowel macrophage regulation). Here, we demonstrate that PELATON is highly monocyte- and macrophage-specific across vascular cell types, and almost entirely nuclear by cellular fractionation (90%-98%). In situ hybridization confirmed enrichment of PELATON in areas of plaque inflammation, colocalizing with macrophages around the shoulders and necrotic core of human plaque sections. Consistent with its nuclear localization, and despite containing a predicted open reading frame, PELATON did not demonstrate any protein-coding potential in vitro. Functionally, knockdown of PELATON significantly reduced phagocytosis, lipid uptake and reactive oxygen species production in high-content analysis, with a significant reduction in phagocytosis independently validated. Furthermore, CD36, a key mediator of phagocytic oxLDL (oxidized low-density lipoprotein) uptake was significantly reduced with PELATON knockdown. CONCLUSIONS: PELATON is a nuclear expressed, monocyte- and macrophage-specific lncRNA, upregulated in unstable atherosclerotic plaque. Knockdown of PELATON affects cellular functions associated with plaque progression.


Assuntos
Artérias Carótidas/metabolismo , Doenças das Artérias Carótidas/metabolismo , Macrófagos/metabolismo , Placa Aterosclerótica , RNA Longo não Codificante/metabolismo , Idoso , Idoso de 80 Anos ou mais , Antígenos CD36/genética , Antígenos CD36/metabolismo , Artérias Carótidas/patologia , Doenças das Artérias Carótidas/genética , Doenças das Artérias Carótidas/patologia , Células Cultivadas , Feminino , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Metabolismo dos Lipídeos , Macrófagos/patologia , Masculino , Necrose , Fagocitose , RNA Longo não Codificante/genética , Espécies Reativas de Oxigênio/metabolismo , Ruptura Espontânea
4.
Int J Mol Sci ; 22(3)2021 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-33540814

RESUMO

Vascular smooth muscle cells (VSMCs) provide vital contractile force within blood vessel walls, yet can also propagate cardiovascular pathologies through proliferative and pro-inflammatory activities. Such phenotypes are driven, in part, by the diverse effects of long non-coding RNAs (lncRNAs) on gene expression. However, lncRNA characterisation in VSMCs in pathological states is hampered by incomplete lncRNA representation in reference annotation. We aimed to improve lncRNA representation in such contexts by assembling non-reference transcripts in RNA sequencing datasets describing VSMCs stimulated in vitro with cytokines, growth factors, or mechanical stress, as well as those isolated from atherosclerotic plaques. All transcripts were then subjected to a rigorous lncRNA prediction pipeline. We substantially improved coverage of lncRNAs responding to pro-mitogenic stimuli, with non-reference lncRNAs contributing 21-32% for each dataset. We also demonstrate non-reference lncRNAs were biased towards enriched expression within VSMCs, and transcription from enhancer sites, suggesting particular relevance to VSMC processes, and the regulation of neighbouring protein-coding genes. Both VSMC-enriched and enhancer-transcribed lncRNAs were large components of lncRNAs responding to pathological stimuli, yet without novel transcript discovery 33-46% of these lncRNAs would remain hidden. Our comprehensive VSMC lncRNA repertoire allows proper prioritisation of candidates for characterisation and exemplifies a strategy to broaden our knowledge of lncRNA across a range of disease states.


Assuntos
Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Placa Aterosclerótica/metabolismo , RNA Longo não Codificante/análise , Aorta/citologia , Vasos Coronários/citologia , Citocinas/farmacologia , Conjuntos de Dados como Assunto , Elementos Facilitadores Genéticos , Perfilação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , RNA Longo não Codificante/isolamento & purificação , RNA-Seq , Estresse Mecânico , Transcrição Gênica/efeitos dos fármacos , Transcriptoma
5.
Circulation ; 133(21): 2050-65, 2016 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-27052414

RESUMO

BACKGROUND: Phenotypic switching of vascular smooth muscle cells from a contractile to a synthetic state is implicated in diverse vascular pathologies, including atherogenesis, plaque stabilization, and neointimal hyperplasia. However, very little is known about the role of long noncoding RNA (lncRNA) during this process. Here, we investigated a role for lncRNAs in vascular smooth muscle cell biology and pathology. METHODS AND RESULTS: Using RNA sequencing, we identified >300 lncRNAs whose expression was altered in human saphenous vein vascular smooth muscle cells following stimulation with interleukin-1α and platelet-derived growth factor. We focused on a novel lncRNA (Ensembl: RP11-94A24.1), which we termed smooth muscle-induced lncRNA enhances replication (SMILR). Following stimulation, SMILR expression was increased in both the nucleus and cytoplasm, and was detected in conditioned media. Furthermore, knockdown of SMILR markedly reduced cell proliferation. Mechanistically, we noted that expression of genes proximal to SMILR was also altered by interleukin-1α/platelet-derived growth factor treatment, and HAS2 expression was reduced by SMILR knockdown. In human samples, we observed increased expression of SMILR in unstable atherosclerotic plaques and detected increased levels in plasma from patients with high plasma C-reactive protein. CONCLUSIONS: These results identify SMILR as a driver of vascular smooth muscle cell proliferation and suggest that modulation of SMILR may be a novel therapeutic strategy to reduce vascular pathologies.


Assuntos
Proliferação de Células/fisiologia , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/fisiologia , RNA Longo não Codificante/fisiologia , Proteínas de Caenorhabditis elegans , Células Cultivadas , Técnicas de Silenciamento de Genes , Humanos , Músculo Liso Vascular/citologia , Veia Safena/citologia , Veia Safena/fisiologia
6.
Mol Ther ; 24(5): 978-90, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26898221

RESUMO

Despite the increasing importance of long noncoding RNA in physiology and disease, their role in endothelial biology remains poorly understood. Growing evidence has highlighted them to be essential regulators of human embryonic stem cell differentiation. SENCR, a vascular-enriched long noncoding RNA, overlaps the Friend Leukemia Integration virus 1 (FLI1) gene, a regulator of endothelial development. Therefore, we wanted to test the hypothesis that SENCR may contribute to mesodermal and endothelial commitment as well as in endothelial function. We thus developed new differentiation protocols allowing generation of endothelial cells from human embryonic stem cells using both directed and hemogenic routes. The expression of SENCR was markedly regulated during endothelial commitment using both protocols. SENCR did not control the pluripotency of pluripotent cells; however its overexpression significantly potentiated early mesodermal and endothelial commitment. In human umbilical endothelial cell (HUVEC), SENCR induced proliferation, migration, and angiogenesis. SENCR expression was altered in vascular tissue and cells derived from patients with critical limb ischemia and premature coronary artery disease compared to controls. Here, we showed that SENCR contributes to the regulation of endothelial differentiation from pluripotent cells and controls the angiogenic capacity of HUVEC. These data give novel insight into the regulatory processes involved in endothelial development and function.


Assuntos
Células Endoteliais/fisiologia , Neovascularização Patológica/genética , RNA Longo não Codificante/genética , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Transdução de Sinais
7.
bioRxiv ; 2023 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-36711681

RESUMO

Background: Activation of vascular smooth muscle cells (VSMCs) inflammation is vital to initiate vascular disease. However, the role of human-specific long noncoding RNAs (lncRNAs) in VSMC inflammation is poorly understood. Methods: Bulk RNA-seq in differentiated human VSMCs revealed a novel human-specific lncRNA called IN flammatory M K L1 I nteracting L ong N oncoding RNA ( INKILN ). INKILN expression was assessed in multiple in vitro and ex vivo models of VSMC phenotypic modulation and human atherosclerosis and abdominal aortic aneurysm (AAA) samples. The transcriptional regulation of INKILN was determined through luciferase reporter system and chromatin immunoprecipitation assay. Both loss- and gain-of-function approaches and multiple RNA-protein and protein-protein interaction assays were utilized to uncover the role of INKILN in VSMC proinflammatory gene program and underlying mechanisms. Bacterial Artificial Chromosome (BAC) transgenic (Tg) mice were utilized to study INKLIN expression and function in ligation injury-induced neointimal formation. Results: INKILN expression is downregulated in contractile VSMCs and induced by human atherosclerosis and abdominal aortic aneurysm. INKILN is transcriptionally activated by the p65 pathway, partially through a predicted NF-κB site within its proximal promoter. INKILN activates the proinflammatory gene expression in cultured human VSMCs and ex vivo cultured vessels. Mechanistically, INKILN physically interacts with and stabilizes MKL1, a key activator of VSMC inflammation through the p65/NF-κB pathway. INKILN depletion blocks ILIß-induced nuclear localization of both p65 and MKL1. Knockdown of INKILN abolishes the physical interaction between p65 and MKL1, and the luciferase activity of an NF-κB reporter. Further, INKILN knockdown enhances MKL1 ubiquitination, likely through the reduced physical interaction with the deubiquitinating enzyme, USP10. INKILN is induced in injured carotid arteries and exacerbates ligation injury-induced neointimal formation in BAC Tg mice. Conclusions: These findings elucidate an important pathway of VSMC inflammation involving an INKILN /MKL1/USP10 regulatory axis. Human BAC Tg mice offer a novel and physiologically relevant approach for investigating human-specific lncRNAs under vascular disease conditions.

8.
Eur J Pharmacol ; 842: 1-9, 2019 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-30359564

RESUMO

In the isolated rat carotid artery, the endocannabinoid anandamide induces endothelium-dependent relaxation via activation of the enzyme sphingosine kinase (SK). This generates sphingosine-1-phosphate (S1P) which can be released from the cell and activates S1P receptors on the endothelium. In anaesthetised mice, anandamide has a well-characterised triphasic effect on blood pressure but the contribution of SK and S1P receptors in mediating changes in blood pressure has never been studied. Therefore, we assessed this in the current study. The peak hypotensive response to 1 and 10 mg/kg anandamide was measured in control C57BL/6 mice and in mice pretreated with selective inhibitors of SK1 (BML-258, also known as SK1-I) or SK2 ((R)-FTY720 methylether (ROMe), a dual SK1/2 inhibitor (SKi) or an S1P1 receptor antagonist (W146). Vasodilator responses to S1P were also studied in isolated mouse aortic rings. The hypotensive response to anandamide was significantly attenuated by BML-258 but not by ROMe. Antagonising S1P1 receptors with W146 completely blocked the fall in systolic but not diastolic blood pressure in response to anandamide. S1P induced vasodilation in denuded aortic rings was blocked by W146 but caused no vasodilation in endothelium-intact rings. This study provides evidence that the SK1/S1P regulatory-axis is necessary for the rapid hypotension induced by anandamide. Generation of S1P in response to anandamide likely activates S1P1 to reduce total peripheral resistance and lower mean arterial pressure. These findings have important implications in our understanding of the hypotensive and cardiovascular actions of cannabinoids.


Assuntos
Anti-Hipertensivos/farmacologia , Ácidos Araquidônicos/farmacologia , Endocanabinoides/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Alcamidas Poli-Insaturadas/farmacologia , Animais , Aorta/efeitos dos fármacos , Aorta/fisiologia , Pressão Sanguínea/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Isoenzimas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Receptores de Lisoesfingolipídeo/metabolismo , Vasodilatação/efeitos dos fármacos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA